Salmonella infections continue to pose a significant threat to human health worldwide. Our studies have established an essential role for the slyA gene in the pathogenesis of Salmonella infections. The SlyA protein belongs to a novel family of low molecular weight transcriptional regulators. SlyA appears to be maximally expressed in stationary phase cultures and in the intracellular environment of phagocytes, slyA mutant Salmonella typhimurium is profoundly attenuated for virulence in a murine model of salmonellosis, unable to survive and replicate within phagocytes, and hypersusceptible to oxidative stress. By DNA microarray analysis, we have identified a number of candidate SlyA-regulated genes. To determine the mechanism by which the SlyA regulon defends S. typhimurium against oxidative stress and contributes to Salmonella pathogenesis, the following Specific Aims of this revised proposal are: (1) Identification and characterization of SlyA-dependent genes. Preliminary experiments have successfully identified a number of candidate SlyA-dependent loci, which will be confirmed by several independent methods (mRNA, protein, reporter fusions). A SlyA-regulated gene in Salmonella Pathogenicity Island-4 designated STM4261 that encodes a large protein with a serine protease motif will be biochemically characterized. STM4261 expression will be measured in wild type and slyA mutant backgrounds, and the virulence of non-polar mutants of STM4261 will be determined. (2) Definition of the role of SlyA-dependent genes in oxidative stress resistance and virulence. The contribution of individual SlyA-dependent loci to oxidative stress resistance, growth in phagocytes, and S. typhimurium virulence will be determined.(3) Molecular analysis of slyA regulation. Transcriptional and translational mechanisms governing slyA expression in S. typhimurium will be determined. Regulatory interactions between SlyA and PhoPQ will be explored. A novel two-component regulatory locus that appears to be essential for slyA expression will be characterized, slyA-dependent promoters will be analyzed, and a consensus binding sequence will be determined. The overall goal of this project is to understand mechanisms by which the SlyA regulon confers resistance to the oxidative stress encountered by Salmonella in host phagocytes. The slyA gene family is conserved among Gram-negative enteric pathogens, as well as several important plant pathogens. Understanding the molecular mechanisms by which the SlyA regulon functions in Salmonella promises to reveal novel mechanisms for intracellular survival of pathogenic bacteria as well as provide important general insights into the evolutionary adaptation of bacteria to oxygen-rich environments. [unreadable] [unreadable]